Program Description/Abstract The majority of cell deaths that occur in the animals do so via apoptosis, and in mammals, this happens predominantly by the mitochondrial pathway. This involves the process of mitochondrial outer membrane permeabilization (MOMP), upon which cytochrome c diffuses to the cytosol, caspase proteases are activated, and apoptosis proceeds. Thus, MOMP is often considered the critical decision point for this active cell death pathway. MOMP is both caused and regulated by proteins of the BCL-2 family, and this control of MOMP is likely to be the most important function of this protein family. This proposal addresses a new unified model for the function of this protein. In this model, anti-apoptotic BCL-2 proteins act either to sequester the proteins that activate MOMP (by activating the effector proteins), or to sequester the active effector proteins themselves. The differences between these two modes of inhibition, and how they can be de-repressed to promote MOMP and apoptosis, are the bases for this application. The following aims will be addressed. 1. Characterize the properties of the two modes of anti-apoptotic BCL-2 protein function and their de- repression. We will employ isolated mitochondria to probe the properties of anti-apoptotic BCL-2 proteins under conditions in which they function by blocking MOMP by sequestration of direct activator proteins (MODE 1) or by sequestration of the effectors, BAX and BAK (MODE 2). The relative efficiencies of the anti-apoptotic proteins will be assessed in terms of BAX/BAK activation and cytochrome c release. We will further examine the relative sensitivity of each MODE to de-repression to induce MOMP. We propose that each condition will display fundamentally different properties, in a manner that cannot be predicted by other models. 2. Analyze the discrete modes of MOMP inhibition by anti-apoptotic BCL-2 proteins in cells. We will then extend our studies into cells to determine if and when these two modes of anti-apoptotic function are engaged upon cell stress. Here, the proposed studies are designed to test and extend our model of BCL-2 family function in controlling MOMP, and establishing conditions under which MODE 1 or MODE 2 predominate in living cells. We will take advantage of biochemical and live cell imaging approaches to follow the behavior of the pro- apoptotic BCL-2 effector proteins in the context of MOMP. 3. Determine the consequences of de- repression of each mode of anti-apoptotic BCL-2 protein function for cell death and survival in cells. Here we will seek to probe the consequences of this model for cell death by de-repression, leading to apoptosis, under defined situations in cells. We will test if MODE 1 versus MODE 2 inhibition is differentially sensitive to de-repression in cells, and how this affects priming for death, observed upon pharmacologic de- repression and/or changes in the functions of anti-apoptotic proteins. These studies provide a number of tests and explorations of the new model we propose, and hold the potential to greatly increase of understanding of this fundamental process controlling life cell and death.